Download Thyroid Hormone Regulation of Peptidylglycine a

Thyroid Hormone Regulation of
Peptidylglycine a-Amidating
Monooxygenase Expression in
Anterior Pituitary Gland
L'Houcine Ouafik, Victor May, David W. Saffen, and
Betty A. Eipper
Department of Neuroscience
Johns Hopkins University School of Medicine
Baltimore, Maryland 21205
Department of Anatomy and Neurobiology
University of Vermont College of Medicine (V.M.)
Burlington, Vermont 05405
a large fraction of the anterior pituitary cell population upon surgical thyroidectomy. These results indicate that thyroid hormones are involved, either
directly or indirectly, in regulating the expression of
PAM in several cell types in the anterior pituitary
gland. (Molecular Endocrinology 4: 1497-1505,
1990)
Peptidylglycine a-amidating monooxygenase (PAM;
EC 1.14.17.3) is a copper-, molecular oxygen-, and
ascorbate-dependent enzyme which catalyzes the
COOH-terminal amidation of bioactive peptides.
Expression of PAM in the adult male rat anterior
pituitary was evaluated after experimental manipulation of thyroid status. Levels of PAM mRNA increased 4- to 7-fold in animals made hypothyroid by
treatment with 6-n-propyl-2-thiouracil or thyroidectomy and were not diminished below control levels
in animals made hyperthyroid by treatment with T4.
Treatment of thyroidectomized animals with T4 prevented the increase in PAM mRNA levels; similar
doses of T4 returned serum TSH and anterior pituitary PAM mRNA to euthyroid values. Based on Northern blot analysis and amplification of fragments derived from rat PAM-1 by reverse transcription and
the polymerase chain reaction, thyroid status did not
affect the distribution of PAM mRNA among its various alternatively spliced forms. The specific activity
of PAM in the anterior pituitary was increased
slightly in both the soluble and particulate fractions
from chemically hypothyroid rats; the majority of the
PAM activity in the rat anterior pituitary was soluble,
and increased secretion of enzyme may account for
the lesser effect of chemical thyroidectomy on specific activity compared to mRNA levels. Western blot
analysis demonstrated a 104-kDa PAM protein in
particulate fractions prepared from control, PTUtreated, and T4-treated animals. The soluble fraction
contained major PAM proteins of 95 and 75 kDa, and
PTU treatment brought about an increase in the
prevalence of the 75-kDa form of PAM protein. In
situ hybridization studies using 35S-labeled fulllength RNA antisense transcripts of rat PAM-1 cDNA
demonstrated an increase in levels of PAM mRNA in
INTRODUCTION
Peptidylglycine a-amidating monooxygenase (PAM; EC
1.14.17.3) is an essential posttranslational processing
enzyme in the biosynthesis of a-amidated peptides (1,
2). PAM produces a-amidated peptide products from a
variety of glycine-extended peptide substrates in a copper-, molecular oxygen-, and ascorbate-dependent
manner. Complementary DNAs encoding PAM have
been isolated from bovine intermediate pituitary, rat
heart atrium, and frog skin libraries (3-6). The three
species express mRNAs encoding PAM precursor proteins of approximately 100 kDa with an amino-terminal
signal sequence, a large intragranular catalytic domain,
a hydrophobic transmembrane domain, and a short
cytoplasmic tail. PAM RNA transcripts undergo tissuespecific and developmentally regulated alternative splicing (4, 7). Soluble and membrane-associated PAM activities have been identified, and their distribution is
tissue specific (8). Studies with purified PAM and PAM
produced by AtT-20 mouse corticotropic tumor cells
transfected with cDNA encoding bovine PAM indicate
that a single enzyme can a-amidate a wide variety of
amino acids (1, 2, 9, 10). Levels of amidation activity
and PAM mRNA are regulated in a tissue-specific fashion in response to various drug treatments and endocrine manipulations both in vivo and in vitro (2, 11).
0888-8809/90/1497-1505$02.00/0
Molecular Endocrinology
Copyright © 1990 by The Endocrine Society
Although the major anterior pituitary hormones are
not a-amidated, PAM levels in the anterior pituitary
1497
Vol4No. 10
MOL ENDO-1990
1498
gland are among the highest in rat tissues (12, 13).
Several a-amidated peptides, including substance-P,
neuropeptide-Y, and vasoactive intestinal peptide (VIP),
have been identified in the rat anterior pituitary gland
(14-16). Hypothyroidism has been shown to greatly
increase the expression of each of these a-amidated
peptides in the anterior pituitary gland (16-18). Previous
studies demonstrated an effect of thyroidectomy on
levels of PAM activity in serum and soluble PAM activity
in the anterior pituitary (2). Although the effects of
thyroid hormones on many cellular metabolic processes
have been well documented (19, 20), little is known
about the effects of thyroid hormones on peptide-processing enzymes. In the present study we have used
several techniques to examine the effects of thyroid
status on PAM expression in the anterior pituitary gland.
Levels of PAM mRNA were evaluated using electrophoretic blot hybridization analysis, and changes in PAM
mRNA forms were investigated using reverse transcription, followed by the polymerase chain reaction. Tissue
levels of PAM activity were measured, and PAM protein
forms were examined by Western blot analysis. In situ
hybridization studies were conducted to determine
whether changes in thyroid hormone status altered
PAM mRNA levels in all or only a fraction of the total
cell population of the anterior pituitary gland.
RESULTS
Regulation of PAM Expression by Thyroid Status
In the first experimental paradigm, adult male rats were
made hypothyroid by treatment with 6-n-propyl-2-thiouracil (PTU) or hyperthyroid by treatment with L-T4 (T4).
The effectiveness of the treatment was verified by
measurement of serum TSH levels (Fig. 1B). After treatment with T4, serum TSH levels were reduced compared to those in vehicle-injected control animals; similarly, serum TSH levels were elevated over control
values in the PTU-treated hypothyroid rats. Anterior
pituitary PAM expression in hyperthyroid and hypothyroid rats was assessed by Northern blot analysis to
examine PAM mRNA levels and forms and by measurement of PAM activity. Total RNA prepared from the
pituitaries of individual rats was subjected to Northern
blot analysis, and PAM mRNA was visualized using a
radiolabeled fragment derived from the 5' region of rat
PAM-1 (rPAM-1) cDNA and capable of detecting all of
the known forms of rat PAM mRNA (Fig. 1A). Pituitary
PAM mRNA ranged from 3.6-3.8 kilobases in size, and
the size distribution was unaltered by thyroid status.
The PAM cDNA probe was removed from the blots,
and the amount of ribosomal RNA present in each
sample was determined by hybridization to a cDNA
probe for ribosomal RNA (Fig. 1 A). The amount of PAM
mRNA in each sample was then normalized to the
amount of ribosomal RNA (Fig. 1B). Anterior pituitary
PAM mRNA levels in the hypothyroid rats were increased 6.7 ± 0.6-fold over control values (n = 3; mean
CONTROL
PTU
PAM
18S
tf f iw^ *
0.8
•12.0
0.4
0.0
Con
PTU
Fig. 1. Effect of Thyroid Status on Expression of PAM mRNA
in Anterior Pituitary
A, Total RNA (10 ng) from pituitaries of individual euthyroid
(control), hyperthyroid (T4), and hypothyroid (PTU) rats was
fractionated on a denaturing 1 % agarose gel and transferred
to Nytran. The blot was hybridized with a full-length rPAM-1
cDNA probe and exposed to x-ray film for 24 h at - 7 0 C with
an intensifying screen. The blot was subsequently stripped
and hybridized with the ribosomal RNA probe (18S). B, After
densitization of the autoradiograms using computer-assisted
densitometry and correction for nonlinearity of film grain density, levels of PAM mRNA were normalized to levels of ribosomal RNA on the same blot; this arbitrary ratio was used to
express relative tissue PAM mRNA levels (•). Serum TSH
levels (H) were assayed for each experimental animal, as
described in Materials and Methods. Error bars indicate the
SD. Stars indicate that the value is significantly different from
the control (•, P < 0.05; • * , P < 0.005). Similar data were
obtained in two additional independent experiments.
± SEM). There was no significant difference in anterior
pituitary PAM mRNA levels between hyperthyroid and
control animals.
Since thyroid status had a dramatic effect on levels
of PAM mRNA, the effect of thyroid status on anterior
pituitary PAM specific activity was also assessed (Fig.
2). Treatment with PTU increased total PAM specific
activity by approximately 60%, while treatment with T4
brought about a slight decrease in the specific activity
of PAM in the anterior pituitary gland. The majority of
the PAM activity (80-90%) was recovered in the soluble
fraction, independent of thyroid status, and the specific
activity of the soluble and particulate fractions mirrored
the total specific activity (Fig. 2). The change in PAM
specific activity after PTU treatment was in the same
direction as the change in levels of PAM mRNA, but
the magnitude of the change in activity was much
smaller. This discrepancy could reflect increased secretion of PAM from the tissue or an alteration in the PAM
protein present. Serum levels of PAM activity are in-
Thyroid Hormone Regulation of PAM Expression
1499
A
B
T4 CON PTU
T4 CON PTU
—116 —
104 —
— 97 —
m
— 66 —
-i til •1
—104
. — 95
|— 84
— 75
—45-
CONTROL
PTU
Fig. 2. Effect of Thyroid Status on PAM Specific Activity in
Anterior Pituitary
PAM specific activity in crude soluble (H) and paniculate (•)
fractions prepared from two pooled anterior pituitary glands
was measured as described in Materials and Methods. Total
PAM specific activity (•) was calculated by taking into account
the amount of protein in the two fractions. Data from two
separate experiments (n = 4 for each treatment group in both
experiments) were used to calculate the mean specific activity;
each sample was assayed in duplicate. Error bars indicate the
SEM. The asterisk indicates that the value is significantly different from the control (P < 0.05).
creased in hypothyroidism (21), although the mechanism responsible for this increase has not been elucidated.
To determine whether the forms of PAM protein
present in the anterior pituitary were affected by thyroid
status, equal amounts of protein prepared from the
soluble and washed particulate fractions from control,
PTU-treated, and T4-treated animals were fractionated
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to Western blot analysis (Fig.
3). The antiserum used to visualize PAM proteins was
raised to a synthetic peptide [bPAM-(561-579)] located
in the intragranular domain of the bovine PAM precursor
protein and was affinity purified before use. The major
PAM protein in all of the anterior pituitary particulate
fractions had a mass of 104 ± 2 kDa (Fig. 3A) and was
also visualized by antiserum to a synthetic peptide
[bPAM-(288-310)] located in the monooxygenase domain of the PAM precursor protein (data not shown).
The qualitative pattern observed for the particulate
fraction was independent of thyroid status.
Analysis of the soluble fraction revealed the presence
of multiple forms of PAM protein (Fig. 3B). In control
and hyperthyroid animals, soluble PAM proteins of 95
and 75 kDa were predominant, with lesser amounts of
104- and 84-kDa PAM proteins. The same forms of
PAM protein were present in PTU-treated animals, but
the 75-kDa PAM protein was more prevalent. Consistent with the magnitude of the effect of thyroid status
Fig. 3. Western Blot Analysis
Two independent aliquots of particulate protein (5 ^g from
T4-treated animals; 20 nQ from controls and PTU-treated animals; A) or soluble protein (50 ^g; B) prepared from the pooled
pituitaries of two T4-treated, control (CON), or PTU-treated
rats were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PAM proteins were visualized with
affinity-purified antiserum to bPAM-(561 -579) and [125l]proteinA. The migration positions of the mol wt markers are indicated
(center). The mol wt of PAM in the particulate fraction is shown
on the left; the mol wt of the soluble PAM proteins are on the
right.
on PAM specific activity, the soluble protein from PTUtreated animals yielded a somewhat more intense signal
than the same amount of protein from control or T4treated animals. Precise quantitative comparisons cannot yet be made, since the cross-reactivity of the antiserum with the various forms of PAM protein is not yet
known. Proteins of the same mass were visualized with
antisera to the monooxygenase domain of PAM (data
not shown). Many of the PAM mRNAs that have been
characterized encode precursor proteins that appear to
undergo endoproteolytic cleavage to generate the major forms of PAM protein found in tissue extracts (2, 4,
8,13), and the 75-kDa PAM protein is thought to derive
from larger precursor forms of PAM protein by endoproteolytic cleavage.1 Thus, the posttranslational processing of the PAM precursor in the pituitary appears to
be sensitive to thyroid status.
Thyroid Hormone Replacement Studies
In the second experimental paradigm, the effect of
thyroid hormone replacement on PAM expression in
thyroidectomized rats was evaluated. Similar to hypothyroidism induced by PTU treatment, hypothyroidism
caused by surgical thyroidectomy resulted in a 3.7 ±
1.1-fold (n = 2; mean ± range) increase in the level of
PAM mRNA in the anterior pituitary gland. When the
thyroidectomized animals were treated with increasing
doses of T4, serum TSH levels declined from the elevated levels observed in hypothyroid animals to levels
below those in euthyroid animals (Fig. 4B). When total
RNA from the individual animals was fractionated on
1
Eipper, B. A., C. B.-R. Green, D. A. Stoffers, H. T. Keutmann, and L'H. Ouafik, manuscript in preparation.
MOL ENDO-1990
1500
VoUNo. 10
A
THYROXINE(«g)
THX
0.25
r o 4 4
Ii
40
64
PAM
THX
0.25
4.0
16
in the rat (Fig. 5, top) (4, 22). In the atrium, the major
forms of PAM mRNA differ by the presence (rPAM-1,
4.2 kb) and absence (rPAM-2, 3.8 kb) of optional exon
A (4); forms of PAM mRNA lacking all or part of optional
exon B are found in the atrium (22), but are more
prevalent in the pituitary (see Footnote 1). The forms of
PAM mRNA in the pituitary are poorly resolved on
denaturing agarose gels. To determine whether thyroid
status altered the forms of PAM mRNA present in the
anterior pituitary, cDNA was prepared by reverse transcription of RNA prepared from control, thyroidectomized, PTU-treated, and T4-treated animals. Pairs of
oligonucleotides spanning the sequence of rPAM-1
were then used as primers in the polymerase chain
reaction (Fig. 5). These studies were carried out to
provide a comparison of the forms of PAM mRNA
present; internal standards permitting correction for
differing efficiencies of reverse transcription or amplifi-
64
fig T 4
Fig. 4. Effect of Thyroidectomy and T4 Replacement on Anterior Pituitary PAM mRNA Expression
A, Total RNA (10 ng) from individual animals in each treatment group was subjected to Northern blot analysis, as described in Fig. 1A. The blot was hybridized first with the PAM
cDNA probe and then stripped and hybridized with the ribosomal RNA probe. Similar results were observed in two independent experiments. B, Quantitative analysis of the blots
shown in A was performed as described in Fig. 1B. Error bars
indicate the range. Serum TSH levels in individual (n = 4)
animals were measured as described; error bars indicate the
SD. The serum TSH level in euthyroid animals was 1.4 ng/ml.
denaturing agarose gels, treatment with increasing
doses of T4 reduced PAM mRNA levels to control values
(Fig. 4). As after treatment with PTU, no alteration in
the size distribution of PAM mRNA could be discerned.
When the levels of PAM mRNA were densitized and
normalized to levels of ribosomal mRNA in each sample,
the decline in anterior pituitary PAM mRNA expression
was seen to occur over the same range of T4 that
produced a decline in serum TSH to euthyroid levels
(Fig. 4B). Thyroid hormone replacement decreased
PAM mRNA levels maximally 4-fold from the elevated
levels observed in hypothyroid animals; in agreement
with the previous study, treatment with doses of T4
high enough to induce hyperthyroidism failed to reduce
PAM mRNA levels below control values. As observed
previously, levels of PAM activity in the serum of thyroidectomized rats were elevated over control levels
(21).
Effect of Thyroid Hormone Treatment on
Expression of Different Alternatively Spliced Forms
of PAM mRNA
Multiple forms of PAM mRNA thought to arise via
alternative splicing of a single gene have been identified
AUG
STOP
4
A
B
94
-P771 ^_'
To
A
rPAM-1
12 3 4
5/19
B
— 1089
12 3 4 5
4/10
—1514
"1460
—1256
9/21
— 710
Fig. 5. Combined Reverse Transcriptase Polymerase Chain
Reaction Demonstration of Alternatively Spliced Forms of PAM
mRNA
Schematic diagram of rPAM-1 cDNA shows the positions
and orientations of oligonucleotide primers used for amplification. The initiation (AUG) and termination (STOP) codons as
well as the positions of optional exons A and B (H) are shown.
The PCR products obtained with the pairs of primers indicated
on the left were fractionated on agarose gels, as described in
Materials and Methods. The number of basepairs present in
the amplified products derived from appropriate plasmid controls is indicated on the right. With primers 4/10 (which span
optional exons A and B) the 1514-bp product corresponds to
rPAM-2, and the 1256-bp product corresponds to rPAM-3
(22); the 1460-bp fragment derives from a form of PAM mRNA
lacking part of optional exon B (see Footnote 1). Samples in
each of the lanes are from 1) control, 2) T4-treated (40 »g T4/
day), 3) PTU-treated, and 4) thyroidectomized animals. Lane
5 in B is a zero DNA control.
Thyroid Hormone Regulation of PAM Expression
cation were not included in the samples, and the results
do not provide a quantitative comparison of levels of
PAM mRNA in different samples. Amplified fragments
were fractionated on agarose gels and identified by
comparison to fragments amplified from plasmids containing cDNA inserts corresponding to each of the
various types of rPAM mRNA (Fig. 5). The first pair of
oligonucleotides (5/19) spans the region from the 5'
end of rPAM-1 to immediately before optional exon A;
the second pair of oligonucleotides (4/10) spans optional exons A and B; the third pair (9/21) extends from
the 3' end of optional exon B to immediately before the
putative poly(A) addition signal. Based on amplification
using these three sets of primers, thyroid status had
no effect on the alternatively spliced forms of rPAM
mRNA in the anterior pituitary; this observation is consistent with the results of Northern blot analysis, which
failed to demonstrate an alteration in the mol wt pattern
of PAM mRNAs after manipulation of thyroid status.
In Situ Hybridization Studies
1501
of resolution, the increase in grain density appeared
uniform across the entire anterior pituitary gland. The
grain density diminished upon thyroid hormone replacement (Fig. 6, C and D).
To determine the fraction of anterior pituitary cells
exhibiting altered PAM expression in response to thyroid status, the tissue sections were exposed to photographic emulsion for 1 week and examined under
brightfield microscopy (Fig. 7). In the control animals,
cells expressing PAM mRNA were found scattered
throughout the anterior pituitary gland. As observed
previously, a subset of the cells was heavily labeled,
but few cells were completely devoid of grains. This
result is consistent with the immunocytochemical localization of high levels of PAM to a subset of gonadotropes, with moderate levels of PAM in corticotropes
and lower levels in somatotropes and lactotropes (23).
After thyroidectomy, PAM mRNA expression increased
dramatically in a large percentage of the pituitary cells;
the increase in PAM expression was clearly not limited
to the population of anterior pituitary cells exhibiting
The anterior pituitary gland is composed of several
different cell types. To determine whether alterations in
thyroid status affected PAM mRNA expression in a
subset of the cell population, frozen anterior pituitary
tissue from control rats, thyroidectomized rats, and
thyroidectomized rats treated with T4 were prepared
for in situ hybridization studies. Autoradiographic grains
from the antisense PAM probe in the control anterior
pituitary tissue represented basal PAM expression. In
the thyroidectomized animals, the density of the autoradiographic grains increased significantly compared to
that in controls (compare Fig. 6, A and B); at this level
Fig. 6. Effect of Thyroid Hormone on Anterior Pituitary PAM
mRNA Expression by in Situ Hybridization
Anterior pituitary glands from control (A), thyroidectomized
(B), and thyroidectomized rats given either 16 ^9 (C) or 4 ng
(D) T4 replacement were separated from the neurointermediate
lobe of the pituitary and embedded together, cryosectioned,
and processed for in situ hybridization using 35S-labeled antisense PAM probe, as described in Materials and Methods.
The sections were exposed to x-ray film for 3 days; the figure
was printed directly from the autoradiogram. Heavy grain
densities are represented by white areas.
Fig. 7. High Resolution in Situ Hybridization for Anterior Pituitary PAM mRNA
Cryosections from the study described in Fig. 6 were dipped
in photographic emulsion for cellular resolution of autoradiographic grains. After a 1-week exposure period, the tissues
were processed and stained with toluidine blue. Anterior pituitary glands from control (A) and thyroidectomized (B) rats were
photographed in the brightfield mode. Scale bar = 50 ^m.
MOL ENDO-1990
1502
high expression of PAM in the basal or euthyroid state.
Grain density appeared to be increased in most of the
cells of the anterior pituitary gland after thyroidectomy,
with a subset of the cells exhibiting greatly enhanced
grain densities compared to the majority of the cells.
DISCUSSION
These results demonstrate that PAM expression in the
rat anterior pituitary is regulated by thyroid hormone. In
the present study we showed that anterior pituitary
PAM expression increased in hypothyroidism. Total
PAM specific activity increased approximately 60% in
PTU-treated rats, and PAM mRNA levels were elevated
4- and 7-fold in thyroidectomized and PTU-treated rats,
respectively. The amount of PAM activity in pituitary
extracts represents a balance among synthesis, storage, and secretion of the enzyme. Thus, the 7-fold
increase in PAM mRNA levels observed in PTU-treated
hypothyroid rats resulted in only a 60% increase in total
PAM specific activity (Fig. 2). As observed previously
(21), the anterior pituitary glands of surgically thyroidectomized rats exhibited a slight decrease in soluble
(and total) PAM specific activity (data not shown) despite an increase in levels of PAM mRNA (Fig. 4).
Similarly, elevated levels of preprohormone mRNA can
be accompanied by reduced tissue levels of product
peptide if secretion is stimulated sufficiently. Injection
of hypothyroid animals with T4 restored PAM mRNA to
control levels. By in situ hybridization, silver grains
accumulated over the cytoplasm of groups of cells
scattered throughout the anterior pituitary gland of the
hypothyroid animals (Fig. 6, A and B).
These results are interesting in light of the stimulatory
effect of hypothyroidism on the production of many aamidated peptides synthesized in the anterior pituitary
gland. Substance-P content in the anterior pituitary is
markedly enhanced by hypothyroidism, restored to normal by T4 replacement, and reduced markedly below
control levels in hyperthyroidism (16). Furthermore, the
expression of preprotachykinin-A mRNA in the anterior
pituitary gland is increased in hypothyroidism and suppressed below control values by T4 treatment (24). The
effects of thyroidectomy on anterior pituitary neuropeptide-Y and VIP levels are strikingly similar to those
described for substance-P (17,18). It will be of interest
to use anterior pituitary tissue from hypothyroid rats as
a model to study the colocalization of these peptides
with PAM, since some of the peptides are present in
very low abundance under normal physiological conditions. VIP cells, for example, are undetectable in normal
rats, but increase dramatically in number after thyroidectomy (18,25). Coordinate regulation of expression of
PAM and another a-amidated peptide, TRH, was previously observed during development of neonatal rat
pancreas (26).
Paracrine interactions mediated by some of these aamidated peptides may well play a role in the alterations
Vol4No. 10
in pituitary function that occur in hypothyroidism and
hyperthyroidism. Many anterior pituitary cell types, including somatotropes, lactotropes, and thyrotropes,
are known to be responsive to thyroid hormones (2729). The function of the hypothalamic-pituitary-adrenal
axis is also affected by thyroid status, although the
fundamental mechanisms underlying this response are
not clear (30, 31).
In well studied examples, such as regulation of TSH
(27), GH (32), PRL (28), S14 protein (33), malic enzyme
(34), and a-myosin heavy chain (35), thyroid hormone
has been shown to act mainly at the transcriptional
level. It is not yet clear whether the effects of thyroid
hormone on PAM expression in the anterior pituitary
gland are direct or indirect. The control of the cytoplasmic level of PAM mRNA may involve changes in
the rate of specific gene transcription, the rate of processing of the PAM primary transcript, or the stability of
the nuclear or cytoplasmic PAM RNA sequences. Although our measurements reflect steady state levels of
mRNA and do not allow us to distinguish changes in
transcriptional rate from changes in RNA stability, these
results indicate that any diminution in levels of thyroid
hormone below euthyroid levels results in increased
expression of PAM. Our current efforts are directed
toward understanding the precise molecular mechanism for this regulation and the physiological significance of these widespread alterations in PAM expression in the anterior pituitary gland in hypothyroidism.
MATERIALS AND METHODS
Animals and Treatments
Male Sprague-Dawley rats (150-200 g; Holtzman Laboratories, Madison, Wl) were maintained on a standard laboratory
diet. In the first experimental paradigm, hyperthyroidism (n =
8) was induced by daily ip injection of T4 (15 ^g T4/100 g BW;
Sigma Chemical Co., St. Louis, MO) for 15 days. Similarly,
hypothyroidism (n = 8) was induced by daily ip injection of
PTU (1 mg PTU/100 g BW; Sigma Chemical Co.). Both T4 and
PTU were dissolved in 0.1 N NaOH and diluted to the appropriate concentration in normal saline solution. Control rats (n
= 8) received daily ip injections of saline solution for the same
period of time. The complete experiment was replicated independently three times.
In the second experimental paradigm, male Sprague-Dawley rats (200 g) were thyroidectomized 6 weeks before thyroid
hormone replacement. Each group of hypothyroid rats received daily ip injections of T4 (0.25, 1,4,16, 40, or 64 ng; n
= 4 for each group) for 15 days. The experiment was replicated
independently two times; control animals received sham injections of vehicle.
At the end of each experiment, the animals were weighed,
and trunk blood was collected for serum TSH measurements.
Reagents for RIA forTSH/S were kindly provided by the NIDDK.
Individual anterior pituitaries were rapidly removed for determination of PAM activity, preparation of total RNA, or in situ
hybridization studies.
Tissue Preparation and Amidation Assays
Anterior pituitary tissue was homogenized in 20 mM NaTES,
(A/-Tris[hydroxymethyl]methyl-2-aminoethane sulfonic acid),
1503
Thyroid Hormone Regulation of PAM Expression
pH 7.4, and 10 mM mannitol and separated into soluble and
crude particulate fractions, as previously described (7, 8).
Soluble fractions were assayed directly. The crude particulate
fractions were solubilized by resuspension in the same buffer
containing 1 % Triton X-100; after centrifugation for 60 min at
100,000 x g, the supernatants were used to measure solubilized membrane-associated PAM activity. Protein concentrations were determined using the bicinchoninic acid protein
assay reagent (Pierce Chemical Co., Rockford, IL) and BSA
as standard.
Amidation assays were performed in duplicate, essentially
as described previously (7, 13). Unless indicated otherwise,
assays contained 20,000-25,000 cpm mono-[125l]D-Tyr-ValGly, 0.4 HM D-Tyr-Val-Gly, 400 HM ascorbate, 2 HM CUSO 4 ,
catalase (100 Mg/m|). and 2 fig protein in 120 mM NaTES
buffer, pH 8.5. Reaction velocities are generally expressed as
picomoles of product formed per ng protein/h (specific activity).
The sum of the amount of PAM activity in the soluble and
crude particulate fractions (taking into account the amount of
protein in each fraction) represents total PAM activity per
pituitary; normalization to total protein per pituitary yields total
specific activity. The variation between duplicate samples was
less than 5%. The reaction velocities reported are initial velocities, using a concentration of substrate about 10-fold below
the Km of the enzyme for peptide substrate. In general, no
more than 10% of the substrate was converted into product
in the assay.
Western Blot Analysis
Samples were fractionated on slab gels containing 10% acrylamide and 0.25% A/.A/'-methylenebisacrylamide using the
buffer system of Laemmli (36). Proteins were electrophoretically transferred to Immobilon membranes (Millipore Corp.,
Bedford, MA) in 25 mM Tris and 192 mM glycine, pH 8.3,
containing 20% methanol (7). Mol wt was estimated by comparison with protein standards (Sigma Chemical Co.) fractionated in an adjacent lane. Immobilon strips were blocked with
BSA, incubated with affinity-purified antiserum to bPAM[561 579] (Ab69, diluted 1:100), and processed essentially as described previously (7), except that [125l]protein-A (ICN, Costa
Mesa, CA; 106 cpm/ml) was used to visualize the cross-reactive bands on autoradiographic film. An affinity-purified antiserum to bPAM[288-310] (Ab100, diluted 1:300) was used to
confirm the results obtained with Ab69.
RNA Isolation and Northern Blot Analysis
Total RNA was prepared from individual anterior pituitaries
using the acid guanidinium isothiocyanate-phenol-chloroform
procedure (37). RNA was denatured and then electrophoresed
on 1 % agarose gels containing 2.2 M formaldehyde, 20 mM
MOPS, 5 mM sodium acetate, and 1 mM EDTA, pH 7.0, and
transferred to Nytran (Schleicher and Schuell, Keene, NH) by
capillary action in 20 x SSC (3.0 M NaCI and 0.3 M sodium
citrate, pH 7.0). Filters were baked, prehybridized, hybridized,
and washed as previously described (7). The 1.3-kb Pst\/
BamH\ fragment of rat PAM-1 cDNA (basepairs 351-1681)
was labeled with [a-32P]dCTP to a specific activity of 109 cpm/
ng by random prime synthesis (Amersham Corp., Arlington
Heights, IL) and used as probe (106 cpm/ml). Mol wt was
estimated by comparison to a RNA ladder (Bethesda Research
Laboratories, Gaithersburg, MD) fractionated in a parallel lane
and stained with acridine orange or blotted and probed with
labeled wild-type X-DNA. To correct for the actual amount of
RNA applied to each lane, blots were stripped and hybridized
to cDNA probes derived from frog ribosomal RNA (7, 13). For
quantitation, autoradiograms were densitized using a LOATS
RAS-1000 image analysis system (Amersham Corp.). Known
amounts of cDNA probe were applied to a nitrocellulose membrane using a slot blot apparatus, and densitization of this
autoradiogram provided a standard curve for converting integrated optical density into disintegrations per min. The amount
of PAM mRNA (disintegrations per min) in each sample was
then normalized to the amount of rRNA (disintegrations per
min) in that sample and plotted as a ratio of PAM mRNA/
rRNA. Total RNA was also prepared from heart atria and apical
ventricular tissues from control, PTU-treated, and T4-treated
adult male rats and subjected to quantitative Northern blot
analysis. Although preliminary data suggested effects of thyroid status on levels of PAM mRNA in the heart (38), neither
PTU nor T4 treatment brought about a large change in levels
of PAM mRNA in atrial or apical ventricular heart tissues.
Thyroid hormones, however, do appear to affect PAM expression in primary neonatal heart atrial myocyte cultures.2
Combined Reverse Transcriptase Polymerase Chain
Reaction
Total RNA (5 ng) from control, hyperthyroid, and hypothyroid
rats was reverse transcribed into cDNA using 1 ng oligo(dT)12.
18 (Pharmacia LKB Biotechnology, Piscataway, NJ) as primer
in a 20-n\ reaction volume containing 50 mM Tris-HCI (pH 8.0),
50 mM KCI, 5 mM MgCI2, 5 /XM dithiothreitol, 50 ng/m\ BSA,
1.25 fi\ RNasin (Promega Corp., Madison, Wl), 0.5 mM each
of four dNTPs, and 12 U AMV reverse transcriptase (Life
Sciences, St. Petersburg, FL) at 42 C for 60 min. The synthetic
oligonucleotide primers used in the polymerase chain reaction
were all 17-mers. Primers yielding sense cDNA were (all basepair numbers are for rPAM-1) (4) no. 5 (366-382), no. 4
(1359-1375), and no. 9 (3124-3140). Primers yielding antisense cDNA were no. 19 (1455-1439), no. 10 (3188-3172),
and no. 21 (3834-3816). Polymerase chain reactions were
performed in a 50-^1 volume according to Cetus specifications:
10 mM Tris-HCI (pH 8.3; at 25 C), 50 mM KCI, 1.5 mM MgCI2,
0.01% gelatin, 200 MM each of four dNTPs, 1 HM each primer,
cDNA derived from 50-250 ng total RNA, and 1.25 U Amplitaq
DNA polymerase (Perkin Elmer Cetus, Norwalk, CT). Samples
were overlayed with one drop of light mineral oil and subjected
to 25 cycles in a MJ Research Thermal Cycler (MJ Research,
Inc., Cambridge, MA). Cycling parameters were generally as
follows. The initial denaturation step was performed at 94 C
for 4 min; the repeat cycle consisted of annealing at 52 C for
1 min, followed by extention at 72 C for 3 min and denaturation
at 94 C for 1 min. The last extension time was lengthened to
5 min. After thermal cycling, most of the oil was manually
removed, and the remaining oil was extracted with chloroform.
Samples were fractionated on agarose gels in 89 mM Tris, 89
mM boric acid, and 2.5 mM EDTA, pH 8.0. After staining with
ethidium bromide, the gels were photographed. It should be
emphasized that internal standards were not included during
reverse transcription or amplification, and the amplified products can be compared only in a qualitative manner.
In Situ Hybridization
Anterior pituitaries were embedded in Tissue-Tek (Miles Laboratories, Elkhart, IN) and frozen in a dry ice-alcohol slurry
(39). Cryosections (16 fim) of anterior pituitary glands from
experimental and control animals were mounted on subbed
glass slides and prepared for hybridization to RNA probes, as
previously described (39). Radiolabeled riboprobes were prepared using uridine 5'-[a-35S-thio]triphosphate (New England
Nuclear, Wilmington, DE) and T3 or T7 RNA polymerase (Promega Corp.) to synthesize, respectively, full-length RNA sense
and antisense transcripts of rPAM-1 cDNA from plasmid Z6
(4). In situ hybridization was performed, as described by Segal
and Shilo (40), with the following modifications. 35S-Labeled
RNA probes were not denatured with alkali; prehybridization
was usually omitted; hybridization was performed for 24 h in
a moist chamber under unsealed silane-treated coverslips at
56 C, using 0.5-1 x 106 cpm probe for each section. Slides
May, V., and K. M. Braas, unpublished observations.
Vol4No. 10
MOL ENDO-1990
1504
were washed twice in 2 x SSC, incubated for 30 min at 30 C
in 2 x SSC containing 10 ^g/ml RNase-A (Worthington Biochemicals, Freehold, NJ), washed twice for 30 min each time
in 2 x SSC, and finally immersed in water for a few seconds.
Sections were dehydrated by immersion in 100% ethanol, air
dried, and exposed to Kodak film (Eastman Kodak, Rochester,
NY) at 4 C for 3-7 days. For resolution at the cellular level,
sections were exposed to Kodak autoradiography emulsion
(NTB-2) for 1 week at room temperature, developed, and
stained with toluidine blue.
10.
11.
12.
Acknowledgments
We wish to thank Doris A. Staffers for providing the rat PAM
cDNA probes and teaching us the technique of RT-PCR, and
Eileen Katsimpiris for preparing the affinity-purified antisera
and performing the Western blot analyses. We also wish to
thank Dick Mains and Karen Braas for their scientific support.
Receipt of the reagents for the TSH0 RIA from the NIDDK and
the National Hormone and Pituitary Program (University of
Maryland School of Medicine) is gratefully acknowledged.
13.
14.
15.
16.
Received June 11, 1990. Revision received July 18, 1990.
Accepted July 18,1990.
Address requests for reprints to: Dr. Betty A. Eipper, Department of Neuroscience, Johns Hopkins University School
of Medicine, 725 North Wolfe Street, Baltimore, Maryland
21205.
This work was supported by Grants DK-32949 from NIH
and DA-00098 from NIDA (to B.A.E.).
17.
18.
REFERENCES
1. Bradbury AF, Smyth DG 1987 Biosynthesis of the Cterminal amide in peptide hormones. Biosci Rep 7:907916
2. Eipper BA, Mains RE 1988 Peptide a-amidation. Annu
Rev Physiol 50:333-344
3. Eipper BA, Park LP, Dickerson IM, Keutmann HT, Thiele
EA, Rodgriquez H, Schofield PR, Mains RE 1987 Structure of the precursor to an enzyme mediating COOHterminal amidation in peptide biosynthesis. Mol Endocrinol
1:777-790
4. Staffers DA, Green CB-R, Eipper BA 1989 Alternative
splicing generates multiple forms of peptidylglycine aamidating monooxygenase in rat atrium. Proc Natl Acad
Sci USA 86:735-739
5. Mizuno K, Ohsuye K, Wada Y, Fuchimura K, Tanaka S,
Matsuo H 1987 Cloning and sequence of cDNA encoding
a peptide C-terminal a-amidating enzyme from Xenopus
laevis. Biochem Biophys Res Commun 148:546-552
6. Ohsuye K, Kitano K, Wada Y, Fuchimura K, Tanaka S,
Mizuno K, Matsuo H 1988 Cloning of cDNA encoding a
new peptide C-terminal a-amidating enzyme having a
putative membrane spanning domain from Xenopus laevis
skin. Biochem Biophys Res Commun 150:1275-1281
7. Ouafik L"H, May V, Keutmann HT, Eipper BA 1989 Developmental regulation of peptidylglycine a-amidating
monooxygenase (PAM) in rat heart atrium and ventricle.
J Biol Chem 264:5839-5845
8. May V, Cullen El, Braas KM, Eipper BA 1988 Membraneassociated forms of peptidylglycine a-amidating monooxygenase activity in rat pituitary. J Biol Chem 263:75507554
9. Perkins SN, Eipper BA, Mains RE 1990 Stable expression
of full-length and truncated bovine peptidylgylcine a-ami-
19.
20.
21.
22.
23.
24.
25.
26.
27.
dating monooxygenase complementary DNAs in cultured
cells. Mol Endocrinol 4:132-139
Tamburini PP, Jones BN, Consalvo AP, Young SD, Lovato
SJ, Gilligan JP, Wennogle LP, Erion M, Jeng AY 1988
Structure-activity relationships for glycine extended peptides and the a-amidating enzyme derived from medullary
thyroid CA-77 cells. Arch Biochem Biophys 267:623-631
Thiele EA, Marek KL, Eipper BA 1989 Tissue-specific
regulation of peptidyl-glycine a-amidating monooxygenase expression. Endocrinology 125:2279-2288
Sakata J, Mizuno K, Matsuo H 1986 Tissue distribution
and characterization of peptide C-terminal a-amidating
activity in rat. Biochem Biophys Res Commun 140:230236
Braas KM, Staffers DA, Eipper BA, May V 1989 Tissue
specific expression of rat peptidylglycine a-amidating
monooxygenase activity and mRNA. Mol Endocrinol
3:1387-1398
Arnaout MA, Garthwaite TL, Martinson DR, Hagen TC
1986 Vasoactive intestinal polypeptide is synthesized in
anterior pituitary tissue. Endocrinology 119:2052-2057
DePalatis LR, Khorram O, Ho RH, Negro-Vilar A, McCann
SM 1984 Partial characterization of immunoreactive substance P in the rat pituitary gland. Life Sci 34:225-235
Jones PM, Ghatei MA, Steel J, O'Halloran D, Gon G,
Legon S, Burrin JM, Leonhardt U, Polak JM, Bloom SR
1989 Evidence for neuropeptide Y synthesis in the rat
anterior pituitary and the influence of thyroid hormone
status: comparison with vasoactive intestinal peptide,
substance P, and neurotensin. Endocrinology 125:334341
Aronin N, Coslovsky R, Chase K 1988 Hypothyroidism
increases substance P concentrations in the heterotopic
anterior pituitary. Endocrinology 122:2911-2914
Lam KSL, Lechan RM, Minamitani N, Segerson TP, Reichlin S 1989 Vasoactive intestinal peptide in the anterior
pituitary is increased in hypothyroidism. Endocrinology
124:1077-1084
Oppenheimer JH, Schwartz HL, Mariash CN, Kinlaw WB,
Wong NCW, Freake HC 1987 Advances in our understanding of thyroid hormone action at the cellular level.
Endocr Rev 8:288-308
Samuels HH, Forman BM, Horowitz ZD, Ye Z-S 1988
Regulation of gene expression by thyroid hormone. J Clin
Invest 81:957-967
Mains RE, Myers AC, Eipper BA 1985 Hormonal, drug,
and dietary factors affecting peptidyl glycine a-amidating
monooxygenase activity in various tissues of the adult
male rat. Endocrinology 116:2505-2515
Staffers DA, Eipper BA, 1989 Multiple forms of rat peptidylglycine a-amidating monooxygenase. In: Rotundo RL,
Ahmad F, Bialy H, Black S, Brew K, Chaitin MH, Glaser
L, Magleby KL, Neary JT, Van Brunt J, Whelan WJ (eds)
ICSU Short Report 9, Advances in Gene Technology:
Molecular Biology and Neuropharmacology. IRL Press,
Oxford, p 120
May V, Ouafik L'H, Eipper BA, Braas KM 1990 Immunocytochemical and in situ hybridization studies of peptidylglycine a-amidating monooxygenase (PAM) in pituitary
gland. Endocrinology 127:358-364
Jonassen JA, Mullikin-Kilpatrick D, McAdam A, Leeman
SE 1987 Thyroid hormone status regulates preprotachykinin-A gene expression in male rat anterior pituitary.
Endocrinology 121:1555-1561
Segerson TP, Lam KSL, Cacicedo L, Minamitani N, Fink
JS, Lechan RM, Reichlin S 1989 Thyroid hormone regulates vasoactive intestinal peptide (VIP) mRNA levels in
the rat anterior pituitary gland. Endocrinology 125:22212223
Ouafik L'H, Giraud P, Salers P, Dutour A, Castanas E,
Boudouresque F, Oliver C1987 Evidence for high peptide
a-amidating activity in the pancreas from neonatal rats.
Proc Natl Acad Sci USA 84:261-264
Shupnik MA, Chin WW, Habener JF, Ridgway EC 1985
1505
Thyroid Hormone Regulation of PAM Expression
28.
29.
30.
31.
32.
33.
34.
Transcriptional regulation of the thyrotropin subunit genes
by thyroid hormone. J Biol Chem 260:2900-2903
Day RN, Maurer RA 1989 Thyroid hormone-responsive
elements of the prolactin gene: evidence for both positive
and negative regulation. Mol Endocrinol 3:931-938
Samuels MH, Wierman ME, Wang C, Ridgway EC 1989
The effect of altered thyroid status on pituitary hormone
messenger ribonucleic acid concentrations in the rat. Endocrinology 124:2277-2282
Kamilaris TC, DeBold CR, Pavlou SN, Island DP, Hoursanidis A, Orth DN 1987 Effect of altered thyroid hormone
levels on hypothalamic-pituitary-adrenal function. J Clin
Endocrinol Metab 65:994-999
Sanchez-Franco F, Fernandez L, Fernandez G, Cacicedo
L1989 Thyroid hormone action on ACTH secretion. Horm
Metab Res 21:550-552
Ye Z-S, Forman BM, Aranda A, Pascula A, Park H-Y,
Casanova J, Samuels HH 1988 Rat growth hormone gene
expression. J Biol Chem 263:7821-7829
Jump DB 1989 Rapid induction of rat liver S14 gene
transcription by thyroid hormone. J Biol Chem 264:46984703
Song M-KH, Dozin B, Grieco D, Rail JE, Nikodem VM
35.
36.
37.
38.
39.
40.
1988 Transcriptional activation and stabilization of malic
enzyme mRNA precursor by thyroid hormone. J Biol
Chem 263:17970-17974
Izumo S, Mahdavi V 1988 Thyroid hormone receptor a
isoforms generated by alternative splicing differentially
activate myosin HC gene transcription. Nature 334:539542
Laemmli UK 1970 Cleavage of structural proteins during
assembly of the head of bacteriophage T4. Nature (Lond)
227:680-685
Chomczynski P, Sacchi N 1987 Single-step method of
RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction. Anal Biochem 162:156-159
May V, Ouafik L'H, Eipper BA, Thyroid hormone regulation
of peptidylglycine a-amidating monooxygenase (PAM)
expression. 71st Annual Meeting of The Endocrine Society, Seattle WA, 1989, p 90 (Abstract 272)
Saffen DW, Cole AJ, Worley PF, Christy BA, Ryder K,
Baraban JM 1988 Convulsant-induced increase in transcription factor messenger RNAs in rat brain. Proc Natl
Acad Sci USA 85:7795-7799
Segal D, Shilo BZ 1986 Tissue localization of Drosophila
melanogaster ras transcripts during development. Mol Cell
Biol 6:2241-2248